Identification of a dynamic gene regulatory network required for pluripotency factor-induced reprogramming of mouse fibroblasts and hepatocytes.

The generation of induced pluripotent stem cells (iPSCs) from somatic cells provides an excellent model to study mechanisms of transcription factor-induced global alterations of the epigenome and genome function. Here, we have investigated the early transcriptional events of cellular reprogramming triggered by the co-expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) in mouse embryonic fibroblasts (MEFs) and mouse hepatocytes (mHeps). In this analysis, we identified a gene regulatory network composed of nine transcriptional regulators (9TR; Cbfa2t3, Gli2, Irf6, Nanog, Ovol1, Rcan1, Taf1c, Tead4, and Tfap4), which are directly targeted by OSKM, in vivo. Functional studies using single and double shRNA knockdowns of any of these factors caused disruption of the network and dramatic reductions in reprogramming efficiency, indicating that this network is essential for the induction and establishment of pluripotency. We demonstrate that the stochastic co-expression of 9TR network components occurs in a remarkably small number of cells, approximating the percentage of terminally reprogrammed cells as a result of dynamic molecular events. Thus, the early DNA-binding patterns of OSKM and the subsequent probabilistic co-expression of essential 9TR components in subpopulations of cells undergoing reprogramming steer the reconstruction of a gene regulatory network marking the transition to pluripotency.

Genomic analysis reveals a novel nuclear factor-κB (NF-κB)-binding site in Alu-repetitive elements.

The transcription factor NF-κB is a critical regulator of immune responses. To determine how NF-κB builds transcriptional control networks, we need to obtain a topographic map of the factor bound to the genome and correlate it with global gene expression. We used a ChIP cloning technique and identified novel NF-κB target genes in response to virus infection. We discovered that most of the NF-κB-bound genomic sites deviate from the consensus and are located away from conventional promoter regions. Remarkably, we identified a novel abundant NF-κB-binding site residing in specialized Alu-repetitive elements having the potential for long range transcription regulation, thus suggesting that in addition to its known role, NF-κB has a primate-specific function and a role in human evolution. By combining these data with global gene expression profiling of virus-infected cells, we found that most of the sites bound by NF-κB in the human genome do not correlate with changes in gene expression of the nearby genes and they do not appear to function in the context of synthetic promoters. These results demonstrate that repetitive elements interspersed in the human genome function as common target sites for transcription factors and may play an important role in expanding the repertoire of binding sites to engage new genes into regulatory networks.

Functional studies of shaggy/glycogen synthase kinase 3 phosphorylation sites in Drosophila melanogaster.

Early studies of glycogen synthase kinase 3 (GSK-3) in mammalian systems focused on its pivotal role in glycogen metabolism and insulin-mediated signaling. It is now recognized that GSK-3 is central to a number of diverse signaling systems. Here, we show that the major form of the kinase Shaggy (Sgg), the GSK-3 fly ortholog, is negatively regulated during insulin-like/phosphatidylinositol 3-kinase (PI3K) signaling in vivo. Since genetic studies of Drosophila melanogaster had previously shown that Wingless (Wg) signaling also acts to antagonize Sgg, we investigate how the kinase might integrate, or else discriminate, signaling inputs by Wg and insulin. Using Drosophila cell line assays, we found, in contrast to previous reports, that Wg induces accumulation of its transducer Armadillo (Arm)/beta-catenin without significant alteration of global Sgg-specific activity. In agreement with a previous study using human GSK-3beta, Wg did not cause phosphorylation changes of the Ser9 or Tyr214 regulatory phosphorylated sites of Sgg. Conversely, as shown in mammalian systems, insulin-induced inhibition of Sgg-specific activity by phosphorylation at the N-terminal pseudosubstrate site (Ser9) did not induce Arm/beta-catenin accumulation, showing selectivity in response to the different signaling pathways. Interestingly, a minigene bearing a Ser9-to-Ala change rescued mutant sgg without causing abnormal development, suggesting that the regulation of Sgg via the inhibitory pseudosubstrate domain is dispensable for many aspects of its function. Our studies of Drosophila show that Wg and insulin or PI3K pathways do not converge on Sgg but that they exhibit cross-regulatory interactions.

Mobile-phone radiation-induced perturbation of gene-expression profiling, redox equilibrium and sporadic-apoptosis control in the ovary of Drosophila melanogaster.

The daily use by people of wireless communication devices has increased exponentially in the last decade, begetting concerns regarding its potential health hazards. Drosophila melanogaster four days-old adult female flies were exposed for 30 min to radiation emitted by a commercial mobile phone at a SAR of 0.15 W/kg and a SAE of 270 J/kg. ROS levels and apoptotic follicles were assayed in parallel with a genome-wide microarrays analysis. ROS cellular contents were found to increase by 1.6-fold (x), immediately after the end of exposure, in follicles of pre-choriogenic stages (germarium - stage 10), while sporadically generated apoptotic follicles (germarium 2b and stages 7-9) presented with an averaged 2x upregulation in their sub-population mass, 4 h after fly's irradiation with mobile device. Microarray analysis revealed 168 genes being differentially expressed, 2 h post-exposure, in response to radiofrequency (RF) electromagnetic field-radiation exposure (≥1.25x, P < 0.05) and associated with multiple and critical biological processes, such as basic metabolism and cellular subroutines related to stress response and apoptotic death. Exposure of adult flies to mobile-phone radiation for 30 min has an immediate impact on ROS production in animal's ovary, which seems to cause a global, systemic and non-targeted transcriptional reprogramming of gene expression, 2 h post-exposure, being finally followed by induction of apoptosis 4 h after the end of exposure. Conclusively, this unique type of pulsed radiation, mainly being derived from daily used mobile phones, seems capable of mobilizing critical cytopathic mechanisms, and altering fundamental genetic programs and networks in D. melanogaster.